Many human diseases are caused by the loss of protein function due to defects or an insufficient amount of a protein. To treat such diseases, related proteins have been directly administered to patients. However, many physiologically active proteins used as medicines are easily degraded in serum before they arrive at target tissues and act therein. For this reason, most physiologically active proteins having therapeutic value are excessively or frequently administered to patients to maintain an appropriate concentration capable of offering satisfactory therapeutic effects.
An approach to solve the above problems is to conjugate with polyethylene glycol (PEGylation) or microencapsulate physiologically active proteins. However, these methods are cumbersome because target proteins are primarily produced in microorganisms and purified, and are then PEGlyated or microencapsulated. In addition, cross-linking may occur at undesired positions, which may negatively affect the homogeneity of final products.
Another approach involves glycosylation. Cell surface proteins and secretory proteins produced by eukaryotic cells are modified by a glycosylation process. Glycosylation is known to influence in vivo stability and function of proteins, as well as their physiological properties. However, since glycosylated proteins can be produced only by eukaryotic cells capable of performing glycosylation, their production process is complicated, and it is difficult to attain homogeneous final products which are glycosylated at all desired positions.
In addition, the conventional techniques all improve the problems associated with administration frequency, but do not increase the physiological efficacy of proteins, leading to excessive dosage. For example, NESP developed by the Amgen Company (see U.S. Pat. No. 6,586,398) improves the frequent administration by extending the half-lives of proteins in the blood, but does not increase the efficacy of proteins, leading to excessive dosage that may induce the production of blocking antibodies.
An approach used to improve the efficacy of physiologically active proteins is to mutagenize some amino acid residues of a wild-type protein to improve biological activity of the protein. Related protein variants are disclosed in the following patent publications: (1) U.S. Pat No. 5,457,089: human erytropoietin (EPO) variants where the carboxyl terminal region was altered to increase binding affinity of EPO to its receptor, (2) International Pat. Publication No. 02/077034: human granulocyte colony stimulating factor (G-CSF) variants where a T-cell epitope was altered to reduce immunogenicity of human G-CSF in humans; (3) International Pat. Publication No. 99/57147: human thrombopoietin (TPO) variants prepared by substituting glutamic acid at the 115 position with lysine, arginine or tyrosine in a TOP protein having an amino acid sequence corresponding to 7th to 151st amino acid residues of human mature TPO; and (4) U.S. Pat. Nos. 6,136,563 and 6,022,711 that disclose human growth hormone variants having alanine substitutions at the 18, 22, 25, 26, 29, 65, 168 and 174 positions.
However, the aforementioned protein variants are altered forms made for improving only therapeutic efficacy regardless of changes in in vivo antigenicity. Thus, the scale, degree and position of these alterations have high potential to induce immune responses in humans. Antigenicity in humans may cause serious adverse effects (Casadevall et al. N. Eng. J. Med. 2002, vol. 346, p. 469).